Abstract
The current study is dedicated to the taxonomy of the genus Phylacia (Hypoxylaceae) in Argentina. Fieldwork in the north of the country provided several fresh collections that were studied, using a polyphasic approach. The secondary metabolite profiles of the specimens were generated by high-performance liquid chromatography hyphenated by diode array and mass spectrometry (HPLC–DAD/MS) of the stromata. This study confirmed the presence of secondary metabolites that are also found in the related genus Daldinia. The detection of binapththalene tetrol (BNT), daldinal B, and daldinol, which are also characteristic of certain species of Daldinia and Hypoxylon, further confirmed the chemotaxonomic affinities within the Hypoxylaceae. The phylogenetic affinities of several species were determined using a multi-gene genealogy based on ITS, LSU, TUB2, and RPB2 sequences, confirming that Phylacia is most closely related to Daldinia, Rhopalostroma, and Thamnomyces. The new species P. lobulata, which features a rather unique stromatal morphology and seems to exhibit apparent host specificity for the endemic tree Pseudobombax argentinum, is described.
Similar content being viewed by others
Introduction
Phylacia Lev. constitutes a small genus of the Hypoxylaceae (Xylariales), which currently includes 12 species (Wijayawardene et al. 2022), which have almost exclusively been reported from the Neotropics. The most recent overview on the taxonomy and biogeography of the genus by Medel et al. (2006) emphasized on material from Mexico. However, this paper did not evaluate type specimens of previously described taxa. Neither did Rodrigues and Samuels (1989), who were the first to describe the anamorphs. These authors classified the conidial stages of three species to be “geniculosporium-like” (sensu Petrini and Petrini 1985), but their Figs. 14 and 15 clearly show that this was a misinterpretation. The conidiogeneous cells shown do not bear any geniculate scars that result from dehiscence of conidia (cf. figure 1C in Petrini and Petrini 1985), but they should rather be classified as nodulisporium-like (sensu Petrini and Petrini 1985—cf. their fig. 1E—as well as in the anamorph classification established by Ju and Rogers 1996). For the taxonomic history of the genus, however, the paper by Rodrigues and Samuels (1989) constitutes a very valuable and complete source. Therefore, we refer to this publication for details and will here only discuss the characteristics of Phylacia in view of modern, polythetic concepts.
Phylacia spp. are characterized by elongated ellipsoid translucent yellow-brownish ascospores arranged in globose, evanescent asci that break up early in development and are arranged in carbonaceous, cleistothecial ascomata, which have no ostiolar canal. The asci lack an apical apparatus, and spores are not actively discharged but released after rupture of the ascomatal apex (Rodrigues and Samuels 1989). These characteristics are highly atypical of the Xylariales, but it has meanwhile been established that the characteristics of the teleomorph are not necessarily in agreement with the phylogeny of this order (Jaklitsch et al. 2016; Wendt et al. 2018; Voglmayr et al. 2022). These modern, polyphasic taxonomic studies showed that in the Xylariales and other groups of Sordariomycetes, the teleomorphic characters have subordinate importance. Anamorphic states as well as chemotaxonomic features often agree better with the molecular phylogeny than the morphology of asci and ascospores. For example, chemotaxonomic studies have revealed great similarities of Phylacia to the genera Daldinia, Rhopalostroma, and Thamnomyces (Bitzer et al. 2008; Stadler et al. 2004, 2010). The stromatal pigments of Phylacia closely resemble those of Daldinia, Thamnomyces, and Rhopalostroma (Stadler et al. 2004). A comparative study using cultures of numerous representatives of the stromatic Xylariales revealed a close chemotaxonomic relationship between the aforementioned genera, as well as Entonaema and Ruwenzoria, which all produced small polyketides like 1,8-naphthol, eutypinol and chromone derivatives, while Hypoxylon species lacked these compounds and produce mellein and isosclerone derivatives instead (Bitzer et al. 2008). This was later corroborated in a polyphasic study by Wendt et al. (2018), where Phylacia was placed in the family Hypoxylaceae, but this phylogeny did not include sequences of Phylacia. Other previous molecular phylogenetic studies that included a few sequences of this genus were only based on the ITS locus. In this study, we wish to fill this gap by creating additional data on Phylacia spp. from Argentina using morphological, chemotaxonomic, and molecular phylogenetic characters.
Experimental
General
All scientific names of fungi follow MycoBank (http://www.mycobank.org). No authorities or years of publication are given beside the taxonomic entries. Names of fungaria and culture collections are abbreviated as recommended in Index Herbariorum (http://sweetgum.nybg.org/science/ih). The chemotaxonomic studies of the stromata were carried out using the same methodology as reported recently by Cedeño-Sanchez et al. (2023).
Samples sources and morphological characterization
The fungal specimens surveyed in this study were collected in the subtropical montane forests of the Argentine Northwest. Microscopical and macroscopical morphology were examined and documented as described by Sir (2021). In addition, PVA-lactophenol was used as a mounting medium to ascertain the presence or absence of the germ slit of the ascospores.
For examination of conidiophores, HPLC profiling and sequencing, cultures of the specimens were obtained from multispore isolates according to Kuhnert et al. (2017). The morphology of cultures was studied as described by Stadler et al. (2014), using phase contrastmicroscopy and differential interference contrast under × 400–1000 optical magnification. Colors of stromatal extracts and cultures were assigned according to Rayner (1970). Cultures designated STMA are stored at interim at the HZI Braunschweig under liquid nitrogen.
DNA extraction, PCR, and molecular phylogenetics
The EZ10 Spin Column Fungal Genomic DNA Mini Preps kit (Bio Basic INC.) was used to extract genomic DNA (gDNA) following the manufacturer’s protocol. For extraction, either hyphal material was removed from a YMA plate with an inoculation loop and transferred to a reaction tube with a screw cap supplied with 5–10 precellys ceramic beads (Preqlab, Germany), or material was taken from a liquid culture containing 30 mL of YM medium, which was incubated at 140 rpm and 23 °C for 2–7 days in a 150-mL Erlenmeyer flask. The samples were homogenized, and the subsequent purification steps were carried out according to the manufacturer’s protocol. Samples were stored at 4 °C until further use.
Sequences of four different DNA loci (internal transcribed spacer, ITS; 28S large subunit of the ribosomal RNA, LSU; second large subunit of the nuclear RNA polymerase, RPB2; β-Tubulin, TUB2) were amplified with PCR with primers as described elsewhere (ITS: ITS1f–ITS4; Gardes and Bruns 1993 and White et al. 1990, respectively; LSU: LR0R–LR7, Vilgalys and Hester 1990; RPB2: fRPB25F/fRPB26F–fRPB27cR, Liu et al. 1999; TUB2: T1/T11–T2/T22, O'Donnell and Cigelnik 1997) and PCR programs as listed in Table 1. PCR products were purified using an EZ-10 Spin Column PCR Product Purification Kit (Bio Basic Inc.) following the manufacturers’ instructions. PCR products were stored at − 20 °C until further use. Sanger DNA sequencing was performed by Microsynth Seqlab GmbH.
Sequences from a forward and a reverse read were processed using Geneious® 7.1.9 (Kearse et al. 2012). The electropherogram was checked for sequencing errors and trimmed manually. For TUB2 and RPB2 derived PCR sequences, sequencing was performed with four (T1, T2, T11, T22) and three different primers (fRPB25F, fRPB27cR, fRPB26F). The sequences were checked for authenticity using the NCBI (Sayers et al. 2022) BLAST® (Basic Local Alignment Search Tool; Altschul et al. 1990) program.
The MAFFT (Multiple Alignment with Fast Fourier Transform; v. 7.017) algorithm implemented in Geneious was used to align each locus separately (Katoh and Standley 2013). The L-INS-i algorithm with a 200PAM/k = 2 scoring matrix, a gap open penalty of 1.53 and an offset value of 0.123 were used. The alignments were automatically curated with gBlocks (Castresana 2000; Castresana 2002; Talavera and Castresana 2007) as implemented in the molecular sequence data management package PhyloSuite v1.2.2 (Zhang et al. 2020).
Molecular phylogenetic trees were inferred using IQTree2 (Minh et al. 2020) following a maximum likelihood and a Bayesian (MrBayes 3.2.7a; Ronquist et al. 2012) approach. The taxon selection followed Wendt et al. (2018) with additional sequences from Sir et al. (2016). Sequence data was retrieved from GenBank (https://www.ncbi.nlm.nih.gov/gene/). An appropriate substitution model was automatically selected by ModelFinder (Kalyaanamoorthy et al. 2017), following the Bayesian information criterion (BIC) for each alignment’s partition (Chernomor et al. 2016) before tree reconstruction with 1000 non-parametric bootstrap replicates (BS, Felsenstein 1985). Additionally, PartitionFinder2 (Lanfear et al. 2016) was used to determine best-fit nucleotide substitution models restricted to the ones available in MrBayes 3.2.7a following the BIC criterion. Options for the Bayesian molecular phylogenetic inference were identical to the settings used by Matio Kemkuignou et al. (2022). The topologies were compared and support values ≥ 50% (BS) or ≥ 0.95 (posterior probability, pp) assigned to the respective bipartitions. Single-gene phylogenetic inferences were carried out following the maximum likelihood criterion with 1000 bootstrap replicates to check for congruency of the resolved Phylacia sequences. Support values were assigned following the previously stated strategy.
Results
Molecular phylogenetic inference
Concurrently, a molecular phylogeny was inferred from a MAFFT aligned and gblocks cured multilocus dataset, which in the end consisted of 421, 1279, 957, and 1143 sites for ITS, LSU, RPB2, and TUB2, respectively (totalling a data matrix of 3800 positions). The topologies resulting from maximum likelihood and Bayesian inference strategies were compared, and support values generated from the Bayesian approach were mapped onto the maximum likelihood tree (lLn = − 38156.2560, Fig. 1). The topologies were rooted against an outgroup consisting of one representative each from the Xylariaceae and the Graphostromataceae (Xylaria, Hypoxylon, and Graphostroma platystomum), compared and found to be identical, with the exception of a polytomy found in the tree inferred from the Bayesian approach in the node featuring the analyzed Phylacia sequences (data not shown) forming a moderately bootstrap supported clade (81% BS).
Sequences of Hypoxylon fragiforme (the generic type) and related taxa assembled to an unresolved clade (H2, 99/1) and with sequences derived from H. lateripigmentum, H. investiens, and H. pulicicidum (H3, 100/1) formed a paraphyletic group. A highly supported clade (92/1) suggested relatedness of the species of H3 with Jackrogersella (J; 100/1) and Annulohypoxylon (A; 98/1), however, with Rostrohypoxylon terebratum (Rho) embedded into Annulohypoxylon with low support (within clade A; 58/0.95). Sequences derived from Pyrenopolyporus formed a highly supported clade (100/1) in a highly supported (88/1) sister position to the daldinoid taxa. Daldinia formed three lineages (D1, D2, and D3), of which the first two were resolved with high statistical support (100/1 each), while relatedness to the third clade D3 was not well supported (60/0.95). Interestingly, sequences derived from D. korfii clustered with sequences derived from E. liquescens and Ruwenzoria pseudoannulata (D2 + E); however, this clade received only low statistical support (55/0.96). Clade D3 clustered basally with high support (99/1) to a clade composed of the Phylacia sequences mentioned earlier, from which sequences derived from Rhopalostroma and Thamnomyces branched off with high and medium support, respectively (Rho, T; 99/1, 72/0.98). Sequences derived from Hypomontagnella (100/1; Hy) clustered with moderate support as sister group to all the former clades (62/0.99). A clade consisting of H. fuscum and allies (93/1; H1) appeared in a basal position (87/1) to this large cluster, to which a clade comprising Parahypoxylon papillatum and Durotheca rogersii (100/1) emerged as sister group (100/1). The resolution of the Phylacia clade was congruent among all single locus phylogenetic inferences (Figs. S1-S4).
Chemotaxonomy
In total, seven methanolic stromatal extracts were analyzed by HPLC–UV-Vis-ESI–MS and evaluated for the occurrence of secondary metabolites. Major constituents, characterized by clearly discernible peaks in the 210 nm trace, were compared with our in-house database of stromatal secondary metabolites described for different representatives of the Hypoxylaceae (data not shown). From a total of eleven discernible peaks relating to compounds, only compounds 2, 6, and 10 could be identified as daldinal B, BNT, and daldinol, respectively, with the help of standards, while 1 resembles entonalactam A. The remainder could not be safely assigned to any of the known metabolites that were previously obtained from stromata of the Hypoxylaceae (cf. Helaly et al. 2018). The compound detection patterns were further assigned to three chemotypes (as summarized in Fig. 3 and Table 2). It would be necessary to collect more material and do destructive preparative work and subsequent NMR spectroscopy to unambigiously identify these yet unknown compounds.
Taxonomic part
Phylacia lobulata Sir & C. Lamb., sp. nov.,
MycoBank N°: 846886 Figs. 1, 2, 3, 4, and 6f − j.
Etymology: The epithet lobulata (Latin lobatis = lobed) refers to stromatal morphology.
Holotype – Argentina, Jujuy province. Dept. Ledesma. Parque Nacional Calilegua, El Pedemontano trail, on dead branches of Pseudobombax argentinum (R.E. Fr.) A. Robyns (“soroche”), 24 May 2015, Sir and Hladki 835 (LIL 159605).
Diagnosis – Differs from all other Phylacia spp. by having lobed stromata and ascospores almost cylindrical with the wall slightly wider on the center of the spores.
Description – Stromata solitary to gregarious, superficial or erumpent, 9 − 22 mm long × 6 − 16 mm diam × 5 − 16 mm thick, irregularly and deeply lobed with a more or less cerebriform pattern, constricted at base, vaguely or definitely stipitate, dark brown to black, surface brown in immature stromata, dark brown with brown spots to black in mature stromata; stromal wall strongly carbonaceous, hard, disintegrating with age in an irregular area to expose the mass of ascospores; with dilute KOH-extractable pigments Greenish Olivaceous (90) after 1 min of incubation. Perithecia cylindrical-tubular 0.9 − 1.1 mm high × 0.2 − 0.4 mm diam, Asci 8 − spored, unitunicate, globose to obovoid, 16 − 29.5 μm long × 13 − 19 μm diam. Ascospores 9.2 − 11.9 (13.1) × 4.0 − 5.9 μm (N = 60, av. 10.9 × 4.7 μm), irregularly arranged, unicellular, pale brown to brown, strongly equilateral, ellipsoid to more or less cylindrical with rounded ends, wall thin, but slightly widening towards the center of the spore 0.5 − 0.6 μm thick, smooth, without germ slit. Conidiogeneous structure on the natural substrate as small powdery green masses at margins or over of young stromata, nodulisporium-like. Conidiophores unbranched or irregularly branched with terminal and intercalary conidiogeneous cells. Conidiophores hyaline to pale brown smooth. Conidiogeneous cells hyaline to pale brown, smooth, 8 − 32 × 1.3 − 2.4 μm, with denticulate conidial secession scars. Conidia globose, hyaline to pale brown, smooth, 2.3 − 2.8 × 1.5 − 2.5 μm.
Culture – Colonies on OA covering Petri dish in 2 weeks, at first whitish becoming Olivaceous Grey (121) velvety to felty, inconspicuously zonate with entire margins, reverse greenish black (124). Sporulation regions at the center, scattered. Conidiogeneous structure identical to that described above from the stroma.
Secondary metabolites – Stromatal extracts contain the tentatively identified entonalactam A (1), daldinal B (2), BNT (6) daldinol (10) and the unknown metabolite 8 (Fig. 2 and Table 3).
Distribution and known host – Phylacia lobulata is restricted to the northernmost area in the Argentine Yungas (Jujuy y Salta province). Frequently, the materials were encountered on dead branches of Pseudobombax argentinum (R.E. Fr.) A. Robyns, “soroche” (Malvaceae), (LIL 159605). Possibly, this fungus is host-specific to this plant.
Additional material studied – Argentina. Jujuy province. Dept. Ledesma. Parque Nacional Calilegua, La Lagunita trail, on dead branches of “soroche,” 26 April 2014, Sir and Hladki 618 (LIL 159606), 645 (LIL 159607); on dead corticated branches, 7 June 2017, Sir 1049 (LIL 159608) 1053 (LIL 159609); same loc., El Pedemontano trail, on dead branches of dicot., 24 May 2015, Sir and Hladki 883 (LIL 159610); Guaraní trail, on dead branches of dicot. 6 June 2017, on dead branches of “sororche,” Sir 1055 (LIL 159611). Salta province. Dept. Orán, road to Islas de Cañas, on dead corticated branches, 29 December 2012, Sir and Hladki 329 (LIL 159612) and 330 (LIL 159613).
Notes – This fungus is clearly a member of Phylacia for its cleistocarpic stomata and deliquescent asci without apical apparatus, originating from geniculate ascogeneous hyphae (Medel et al. 2006). The lobed stromata and the ascospores with thickening walls towards the spore center are the most salient discriminatory features for distinguishing it from all other known species of Phylacia. The latter show cylindrical, hemispherical, pulvinate, clavate, conical, pyriform, subglobose, or turbinate stroma and have ascospores with homogeneously thickened walls (Dennis 1957; Fournier and Lechat 2015; Medel et al. 2006).
Phylacia globosa Lév., Annls Sci. Nat., Bot., sér. 3 3: 61 (1845). Fig. 1, 2, and 6a–e.
For a detailed description, figure, and taxonomic notes, see Daranagama et al. (2018).
Secondary metabolites – Stromatal extracts contain BNT (6) and the unknown metabolites 3, 4, 7, and 11 (Fig. 2 and Table 3).
Materials studied – Argentina, Jujuy province, Dept. Ledesma. Parque Nacional Calilegua, La Junta trail, 6 June 2017, Sir 1050 (LIL 159614), 1054 (LIL159615), 1056 (LIL 159616); same loc., La Lagunita trail, 7 June 2017, Sir 1052 (LIL 159617).
Phylacia surinamensis (Berk. & M.A. Curtis) Dennis, Kew Bull. [12] (2): 325 (1957) Figs. 1, 2, 5, and 6k–o.
Description – Stromata densely caespitose, arise from the same stromal base erumpent or superficial, 2.5 − 5 mm high × 1.5 − 3.5 mm diam, more or less cylindrical to slightly clavate, constricted at center and rosette-like, with blackish carbonous surface, hard; outer crust flattened or with slightly concave apex, disintegrating with age in a defined circular area to expose the mass of ascospores, stromal wall strongly carbonaceous, externally coated by a continuous blackish crust yielding Dull Green (70) to Greenish Olivaceous (90) KOH-extractable pigments after 1 min incubation. Perithecia cylindrical-tubular 0.5 − 0.9 mm high × 0.2 − 0.3 mm diam, numerous, compact. Asci 8-spored, spherical, 14.7 − 18.8 µm diam. Ascospores (11.1) 11.5 − 13.5 (14.2) × (5.4) 5.8 − 7.5 (7.8) μm, (n = 60, av. 12.3 × 6.5 μm), irregularly arranged, unicellular, brown to pale brown ellipsoid, equilateral to slightly inequilateral with a germ slit (detectable in lactophenol) and wall thin, smooth. Conidiogenous structure not observed.
Culture – Colonies on OA covering Petri dish in 2 weeks, at first whitish becoming Olivaceous Grey (121) at the center and with entire and Pure Yellow (14) margin; velvety to felty, reverse greenish black (124). Sporulation not observed.
Secondary metabolites – Stromatal extracts contain the tentatively identified entonalactam A (1), daldinal B (2), BNT (6) daldinol (10) and the unknown metabolite 8 (Fig. 2 and Table 3).
Known distribution and host – Phylacia surinamensis is common in the urban areas of Tucuman province and is also encountered in natural reserves from Jujuy province (Argentina). The fresh stromata of this species have been found on recently dead branches and trunks of Ceiba sp. (Malvaceae). This fungus was previously recorded in Brazil (Amazonas), Guatemala, Mexico, and Surinam (Medel et al. 2006).
Materials studied – Argentina. Jujuy province. Dept. Ledesma, Parque Nacional Calilegua, road to La Lagunita trail, on dead branches of Ceiba sp., 11 May 2012, Sir & Hladki 042 (LIL 159618); same loc., 26 April 2014, on dead branches of Ceiba sp, Sir & Hladki 621 (LIL 159619); 26 May 2015, on dead branches of Ceiba sp., Sir & Hladki 832 (LIL 159620), 833 (LIL 159621); 6 June 2017, Guaraní trail, on dead branches of Ceiba sp., Sir 1051 (LIL 159622); 7 June 2017, on dead branches of Ceiba sp., Sir 1057 (LIL 159623). Tucuman province. Dept. Capital, Parque Avellaneda, 19 Jan 2019, on dead branches of Ceiba sp., Sir 1237 (LIL 159624); same loc., Parque 9 de Julio, 8 June 2019, dead branches of Ceiba sp., Sir 1238 (LIL 159625). GUATEMALA. Uaxantun, on dead Ceiba sp., April 1931, H.H. Bartlett 12443, det. J. H. Miller as Camillea surinamensis – (MICH ex LIL).
Notes – This taxon is characterized by having densely caespitose stromata and by its elliptical ascospores. Its stromata are usually cylindrical with flattened or slightly concave apex and grouped on a broad stromatic base (Dennis 1957). The Argentine materials show stromata cylindrical with slight constrictions at the center; in some cases, their shape can be almost clavate.
Phylacia cylindrica has a similar stromatal shape as P. surinamensis, but these species differ by the colors of KOH-extractable pigments (purple vinaceous vs green) and by their ascospore size (Lacerda et al. 2018). The ascospores in Phylacia taxa are apparently devoid of germ slits; Dennis (1957) however illustrated an ascospore with a notable germ slit for P. surinamensis. Lacerda et al. (2018) studied the type specimen and recognized a conspicuous germ slit in old ascospores of this species. Medel et al. (2006) also mentioned the presence of this feature for a collection of P. sagrana (Mont.) Mont. from Costa Rica. Fournier and Lechat (2015) also reported germ slits in ascospores of three species: P. bomba, P. cf. sagrana, and P. korfii, when the spores were mounted in PVA-lactophenol.
The salient discriminatory features of P. surinamensis, P. globosa, and P. lobulata are summarized and compared in Table 4 and illustrated in Fig. 6.
Discussion
This study reports several new records of Phylacia collected in Argentina that were analyzed by a polyphasic approach using a multigene molecular phylogenetic inference and a chemotaxonomic study of their stromatal constituents by HPLC–DAD-MS. The affinities of the genus to Daldinia and Thamnomyces were already described by Ju et al. (1997), which was later corroborated by a chemotaxonomic study on the type and authentic specimens by Stadler et al. (2004). In that paper, binaphthalene derivatives such as BNT, azaphilones of the daldinin type, and daldinal derivatives were reported from stromatal extracts of P. bomba, P. globosa, P. poculiformis, P. sagraena, P. surinamensis, and P. turbinata. These secondary metabolites are commonly encountered in certain species of Daldinia (D. childiae group; cf. Stadler et al. 2014), but also occur in the Hypoxylon fuscum complex (Stadler et al. 2008b; Lambert et al. 2021). This paper is thus the first to use an integrative approach to the taxonomy of the genus that relies on a significant number of freshly collected specimens.
Another chemotaxonomic study of numerous strains that now belong in the Hypoxylaceae included a culture of Phylacia sagrana (CBS 119992), which produced several small polyketides, present also in the concurrently studied cultures of Daldinia spp., but apparently absent in cultures of Annulohypoxylon, Hypoxylon, and other genera now classified as Jackrogersella or Pyrenopolyporus (Bitzer et al. 2008; see also Wendt et al. 2018 for the current taxonomy of these genera and species). The secondary metabolite profile of this Phylacia culture was most similar to that of D. caldariorum, to which it also showed the closest phylogenetic relationship in an ITS-based phylogenetic tree. A similar pattern was described for a subsequent study focusing on Thamnomyces (Stadler et al. 2010), where an additional species of Phylacia (P. poculiformis) was included. The sequences of the two Thamnomyces spp. and both Phylacia species were shown to resolve within the same phylogenetic clade. The current study confirmed and settled the phylogenetic affinities of the genus using, for the first time for Phylacia, a multi-gene genealogy and resolving its placement inside the Hypoxylaceae. These results confirm previous studies as rDNA-derived sequence information was repeatedly shown to be of questionable utility. For the Hypoxylaceae, it was shown that polymorphisms of the rDNA cistron on the one hand, and high redundancies of ITS sequences across several species complexes occur on the other hand (see Stadler et al. 2020 and Maharachchikumbura et al. 2021 for an extensive discussion of this matter).
Our investigation on chemotaxonomically informative secondary metabolites in the stromatal extracts yielded compounds with five different UV–Vis spectra types. In P. surinamensis Sir 1056, a metabolite (1) occurred whose UV–Vis and mass spectra are reminiscent of the reported spectra for entonalactam A, isolated and reported from the stromata of an Australian fungus assigned to Entonaema sp. by Choomuenwai et al. (2015). However, since the authors did not specify how the fungus was identified and did not deposit a voucher, the identity of the material remains unclear. In the same specimen, three other compounds were found that were not detected in the other Phylacia extracts, of which one compound (2) resembled daldinal B (also isolated by Choomuenwai et al. 2015), while the others (8 and 10) showed a characteristic triple-UV maxima in the shape of a crown, indicative of naphthalene derivatives. The mass (616 Da) of 8 suggests a dimer of 6 (318 Da), but could not be assigned to a known compound, while 10 resembled daldinol. Compound 6 could be assigned to BNT and occurred in all studied specimen. Overall, the spectra of the metabolites of P. surinamensis shared similarities with the ones reported for the holotype by Stadler et al. (2004). The UV–Vis spectrum of compound 3 in P. globosa stromata was reminiscent of daldinin derivatives, like daldinin F, but was much smaller in comparison (346 versus 460 Da for daldinin F). All other compounds (4, 5, 7, 9, and 11) shared a similar UV–Vis pattern, which resembled that of daldinone B, but eluted either at a more hydrophilic (4 and 5) or more lipophilic (7, 9, and 11) mobile phase gradient, respectively. Furthermore, compounds 4, 7, and 11 could only be detected in P. globosa, while 5 and 9 only occurred in P. lobulata.
All in all, comparison with the data of the previous study by Stadler et al. (2004) was difficult because comparative data relied mostly on ancient type specimens. In some of them, artifacts, like obvious degradation products and even compounds that may represent insecticides that were eventually added to the specimens for preservation, were detected. However, results like the detection of daldinals in P. surinamiensis are quite significant as that compound seems to have remained stable not only in the holotype specimen of this species for 150 years (Stadler et al. 2004), but also in several ancient specimens of Daldinia childiae (Stadler et al. 2014).
We did not dispose of sufficient material of the valuable specimens that would have allowed for the isolation of the unknown metabolites by preparative HPLC and confirm their chemical structures by means of nuclear magnetic resonance (NMR) spectroscopy and high-resolution MS. However, we have included retention times, mass ionization patterns, and UV/Vis spectra for all the unknown metabolites in the SI (Supplementary Figs. S1–S11) in a hope that these data can aid in future attempts to accomplish such tasks.
Our study also revealed new phylogenetically relevant evidence because we have included some taxa that were not formerly characterized using the current multi-locus approach. For example, Entonaema liquescens formed a well-supported cluster with D. korfii, a taxon that had not been included in recent phylogenies. This was rather unexpected because of the strongly diverging morphology of the respective taxa. The sequenced Entonaema liquescens culture presumably originated from a specimen collected in Kansas by R. Lichtwardt in 1979 (Rogers 1982; Stadler et al. 2008a). To date, it is the only one available of the genus. It was deposited by Jack D. Rogers in ATCC, following the first report on the anamorph of this genus (Rogers 1982). Concerns regarding the authenticity of this Entonaema strain have already been raised (Wibberg et al. 2021; Kuhnert et al. 2021). It was reported that the genome of the strain showed rather high affinities to that of D. concentrica. However, the biosynthesis gene clusters encoding for mitorubrin type azaphilones, which are omnipresent in the stromata of E. liquescens, were not detected in the genome of the ATCC strain. This phenomenon needs further study and requires authentic cultures of E. liquescens that can be studied for comparison.
Data availability
All additional data (except for the DNA sequence data, which are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/)) are available in the manuscript or the supplementary information.
References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–404. https://doi.org/10.1016/S0022-2836(05)80360-2
Bills GF, Gonzalez-Menendez V, Martin J et al. (2012) Hypoxylon pulicicidum sp. nov. (Ascomycota, Xylariales), a pantropical insecticide-producing endophyte. PLoS One 7:e46687 https://doi.org/10.1371/journal.pone.0046687
Bitzer J, Laessøe T, Fournier J et al (2008) Affinities of Phylacia and the daldinoid Xylariaceae, inferred from chemotypes of cultures and ribosomal DNA sequences. Mycol Res 112:251–270. https://doi.org/10.1016/j.mycres.2007.07.004
Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552. https://doi.org/10.1093/oxfordjournals.molbev.a026334
Castresana J (2002) Estimation of genetic distances from human and mouse introns. Genome Biol 3(research0028):1. https://doi.org/10.1186/gb-2002-3-6-research0028
Cedeño-Sanchez M, Charria-Girón E, Lambert C, Luangsa–ard JJ, Decock C, Franke R, Brönstrup M, Stadler M, (2023) Segregation of the genus Parahypoxylon (Hypoxylaceae) from Hypoxylon by a polyphasic taxonomic approach. MycoKeys 95:131–162. https://doi.org/10.3897/mycokeys.95.98125
Chernomor O, von Haeseler A, Monh BQ (2016) Terrace aware data structure for phylogenomic inference from supermatrices. Syst Biol 65(6):997–1008. https://doi.org/10.1093/sysbio/syw037
Choomuenwai V, Beattie KD, Healy PC, Andrews KT, Fechner N, Davis RA (2015) Entonalactams A-C: isoindolinone derivatives from an Australian rainforest fungus belonging to the genus Entonaema. Phytochemistry 117:10–16. https://doi.org/10.1016/j.phytochem.2015.05.018
Daranagama DA, Camporesi E, Tian Q et al (2015) Anthostomella is polyphyletic comprising several genera in Xylariaceae. Fungal Divers 73:203–238. https://doi.org/10.1007/s13225-015-0329-6
Daranagama DA, Hyde KD, Sir EB et al (2018) Towards a natural classification and backbone tree for Graphostromataceae, Hypoxylaceae, Lopadostomataceae and Xylariaceae. Fungal Divers 88:1–165. https://doi.org/10.1007/s13225-017-0388-y
Dennis RWG (1957) Further notes on tropical American Xylariaceae. Kew Bull 1957:297–332
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791
Fournier J, Lechat C (2015) Phylacia korfii sp. nov., a new species of Phylacia (Xylariaceae) from French Guiana, with notes on three other Phylacia spp. Ascomycete.org, 7(6):315–319. https://doi.org/10.25664/art-0154
Fournier J, Stadler M, Hyde K, Duong L (2010) The new genus Rostrohypoxylon and two new Annulohypoxylon species from Northern Thailand. Fungal Divers 40:23–36. https://doi.org/10.1007/s13225-010-0026-4
Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118
Helaly SE, Thongbai B, Stadler M (2018) Diversity of biologically active secondary metabolites from endophytic and saprotrophic fungi of the ascomycete order Xylariales. Nat Prod Rep 35:992–1014. https://doi.org/10.1039/c8np00010g
Hsieh HM, Ju YM, Rogers JD (2005) Molecular phylogeny of Hypoxylon and closely related genera. Mycologia 97:844–865
Hsieh HM, Lin CR, Fang MJ, Rogers JD, Fournier J, Lechat C, Ju YM (2010) Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Mol Phylogenet Evol 54:957–969. https://doi.org/10.1016/j.ympev.2009.12.015
Jaklitsch WM, Gardiennet A, Voglmayr H (2016) Resolution of morphology-based taxonomic delusions: Acrocordiella, Basiseptospora, Blogiascospora, Clypeosphaeria, Hymenopleella, Lepteutypa, Pseudapiospora, Requienella, Seiridium and Strickeria. Persoonia 37:82–105
Johannesson H, Laessøe T, Stenlid J (2000) Molecular and morphological investigation of the genus Daldinia in Northern Europe. Mycol Res 104:275–280. https://doi.org/10.1017/S0953756299001719
Ju YM, Rogers JD (1996) A revision of the genus Hypoxylon. Mycologia Memoir n° 20. APS Press, St. Paul, p 365
Ju YM, Rogers J, San Martín F (1997) A revision of the genus Daldinia. Mycotaxon 61:243–293
Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Meth 14(6):587–589. https://doi.org/10.1038/nmeth.4285
Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software v. 7: improvements in performance and usability. Mol Biol Evol 30(4):772–780. https://doi.org/10.1093/molbev/mst010
Kearse M, Moir R, Wilson A, Stones–Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics (Oxford, England) 28(12):1647–1649. https://doi.org/10.1093/bioinformatics/bts199
Kuhnert E, Fournier J, Peršoh D, Luangsa–ard JJD, Stadler M (2014) New Hypoxylon species from Martinique and new evidence on the molecular phylogeny of Hypoxylon based on ITS rDNA and β-tubulin data. Fungal Divers 64:181–203.https://doi.org/10.1007/s13225-013-0264-3
Kuhnert E, Sir EB, Lambert C et al (2017) Phylogenetic and chemotaxonomic resolution of the genus Annulohypoxylon (Xylariaceae) including four new species. Fungal Diver 85:1–43. https://doi.org/10.1007/s13225-016-0377-6
Kuhnert E, Navarro-Muñoz JC, Becker K, Stadler M, Collemare J, Cox RJ (2021) Secondary metabolite biosynthetic diversity in the fungal family Hypoxylaceae and Xylaria hypoxylon. Stud Mycol 99:1–43. https://doi.org/10.1016/j.simyco.2021.100118
Lacerda LT, Bezerra JL, Pereira J (2018) Phylacia cylindrica sp. nov. from Brazil. Mycotaxon 133(2):243–247. https://doi.org/10.5248/133.243
Lambert C, Wendt L, Hladki AI, Stadler M, Sir EB (2019) Hypomontagnella (Hypoxylaceae): a new genus segregated from Hypoxylon by a polyphasic taxonomic approach. Mycol Progr 18:187–201. https://doi.org/10.1007/s11557-018-1452-z
Lambert C, Pourmoghaddam MJ, Cedeño-Sanchez M, Surup F, Khodaparast SA, Krisai-Greilhuber I, Voglmayr H, Stradal TEB, Stadler M (2021) Resolution of the Hypoxylon fuscum complex (Hypoxylaceae, Xylariales) and discovery and biological characterization of two of its prominent secondary metabolites. J Fungi 7:131. https://doi.org/10.3390/jof7020131
Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2016) Partition Finder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol Biol Evol 34:772–773. https://doi.org/10.1093/molbev/msw260
Liu Y, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol Biol Evol 16:1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092
Maharachchikumbura SSN, Chen Y, Ariyawansa HA, Hyde KD, Haelewaters D Perera RH, Samarakoon MC, Wanasinghe DN, Bustamante DE, Liu JK, Lawrence DP, Cheewangkoon R, Stadler M (2021) Integrative approaches for species delimitation in Ascomycota. Fungal Divers 109:155–179.https://doi.org/10.1007/s13225-021-00486-6
Matio Kemkuignou B, Schweizer L, Lambert C, Anoumedem EGM, Kouam SF, Stadler M, Marin-Felix Y (2022) New polyketides from the liquid culture of Diaporthe breyniae sp. nov. (Diaporthales, Diaporthaceae). Mycokeys 90:85–118. https://doi.org/10.3897/mycokeys.90.82871
Medel R, Rogers JD, Guzman G (2006) Phylacia mexicana sp. nov. and consideration of other species with emphasis on Mexico. Mycotaxon 97:279–290
Minh BQ, Schmidt HA, Chernomor O et al (2020) IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37:1530–1534. https://doi.org/10.1093/molbev/msaa015
Mirabolfathy M, Ju YM, Hsieh HM, Rogers JD (2012) Obolarina persica sp. nov., associated with dying Quercus in Iran. Mycoscience 54:315–320. https://doi.org/10.1016/j.myc.2012.11.003
Petrini L, Petrini O (1985) Xylariaceous fungi as endophytes. Sydowia 38:216–234
Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute, Kew and British Mycological Society
Rodrigues KF, Samuels GJ (1989) Studies in the genus Phylacia (Xylariaceae). Mem N Y Bot Gard 49:290–297
Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61(3):539–542. https://doi.org/10.1093/sysbio/sys029
Rogers JD (1982) Entonaema liquescens: Description of the anamorph and thoughts on its systematic position. Mycotaxon 15:500–506
O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116. https://doi.org/10.1006/mpev.1996.0376
Sayers EW, Cavanaugh M, Clark K et al (2022) GenBank Nucl Acids Res 50:161–164. https://doi.org/10.1093/nar/gky989
Sir EB (2021) La familia Hypoxylaceae (Xylariales, Ascomycota) en Las Yungas del Noroeste argentino. (1ª Ed.). Fundación Hongos de Argentina para la Sustentabilidad
Sir EB, Lambert C, Wendt L et al (2016) A new species of Daldinia (Xylariaceae) from the Argentine subtropical montane forest. Mycosphere 15:1–19. https://doi.org/10.5943/mycosphere/7/9/11
Sir EB, Becker K, Lambert C, Bills GF, Kuhnert E (2019) Observations on Texas hypoxylons, including two new Hypoxylon species and widespread environmental isolates of the H. croceum complex identified by a polyphasic approach. Mycologia 111(5):832–856. https://doi.org/10.1080/00275514.2019.1637705
Stadler M, Ju YM, Rogers JD (2004) Chemotaxonomy of Entonaema, Rhopalostroma and other Xylariaceae. Mycol Res 108:239–256. https://doi.org/10.1017/s0953756204009347
Stadler M, Fournier J, Læssøe T, Lechat C, Tichy HV, Piepenbring M (2008a) Recognition of hypoxyloid and xylarioid Entonaema species from a comparison of holomorphic morphology, HPLC profiles, and ribosomal DNA sequences. Mycol Progr 7:53–73
Stadler M, Fournier J, Beltrán–Tejera E, Granmo A (2008b) The “red Hypoxylons” of the temperate and subtropical Northern Hemisphere. In “A Festschrift in honor of Professor Jack D. Rogers (Glawe DA, Ammirati JF, eds.). North American Fungi 3:73–125. https://doi.org/10.2509/naf2008.003.0075
Stadler M, Flessa F, Rambold G et al (2010) Chemotaxonomic and phylogenetic studies of Thamnomyces (Xylariaceae). Mycoscience 51:189–207. https://doi.org/10.1007/s10267-009-0028-9
Stadler M, Kuhnert E, Peršoh D, Fournier J (2013) The Xylariaceae as model example for a unified nomenclature following the “One Fungus-One Name” (1F1N) concept. Mycology 4:5–21. https://doi.org/10.1080/21501203.2013.782478
Stadler M, Læssøe T, Fournier J, Decock C, Schmieschek B, Tichy H-V, Peršoh D (2014) A polyphasic taxonomy of Daldinia (Xylariaceae). Stud Mycol 77:1–143. https://doi.org/10.3114/sim0016
Stadler M, Lambert C, Wibberg D et al (2020) Intragenomic polymorphisms in the ITS region of high-quality genomes of the Hypoxylaceae (Xylariales, Ascomycota). Mycol Progr 19:35–245. https://doi.org/10.1007/s11557-019-01552-9
Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56:564–577. https://doi.org/10.1080/10635150701472164
Tang AM, Jeewon R, Hyde KD (2007) Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences. Mycol Res 111:392–402. https://doi.org/10.1016/j.mycres.2007.01.009
Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246. https://doi.org/10.1128/jb.172.8.4238-4246.1990
Voglmayr H, Tello S, Jaklitsch WM et al (2022) About spirals and pores: Xylariaceae with remarkable germ loci. Persoonia 49:58–98. https://doi.org/10.3767/persoonia.2022.49.02
Wendt L, Sir EB, Kuhnert E et al (2018) Resurrection and emendation of the Hypoxylaceae, recognised from a multigene phylogeny of the Xylariales. Mycol Prog 17:115–154. https://doi.org/10.1007/s11557-017-1311-3
Wijayawardene NN, Hyde KD, Dai DQ et al (2022) Outline of fungi and fungus-like taxa – 2021. Mycosphere 13:53–453
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA et al. (eds) PCR protocols: a guide to methods and -applications. Academic Press USA, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
Wibberg D, Stadler M, Lambert C et al (2021) High quality genome sequences of thirteen Hypoxylaceae (Ascomycota) strengthen the phylogenetic family backbone and enable the discovery of new taxa. Fungal Divers 106:7–28. https://doi.org/10.1007/s13225-020-00447-5
Zhang D, Gao F, Jakovlić I et al (2020) PhyloSuite: an integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Mol Ecol Res 20:348–355. https://doi.org/10.1111/1755-0998.13096
Acknowledgements
We thank Sebastian Pfütze for providing HPLC chromatograms of the Phylacia specimens. We want to express our gratitude to Anke Skiba for expert technical assistance in culture preservation. The authors express their appreciation to the authorities of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Fundación Miguel Lillo (FML) for the constant support. The Administración de Parques Nacionales of Argentina, Ministerio de Medio Ambiente of Salta Province and Dirección Provincial de Biodiversidad of Jujuy Province are kindly acknowledged for authorization of collection.
In particular, EBS is thankful to Adriana I. Hladki from FML for her invaluable support during the study of the Hypoxylaceae from Northwestern Argentina.
Funding
Open Access funding enabled and organized by Projekt DEAL. This work was funded by the DFG (Deutsche Forschungsgemeinschaft) priority program “Taxon-Omics: New Approaches for Discovering and Naming Biodiversity” (SPP 1991). We are also grateful to the LifeScience Foundation (Munich) for a PhD stipend for C.L.
The German Academic Exchange service (DAAD) and the Ministerio de Ciencia, Tecnología e Innovación, Argentina (MINCyT), are thanked for an academic exchange grant (DAAD-PPP 57052123/MINCyT DA/13/03; Project titles: Taxonomie, Phylogenie und funktionelle Biodiversität neotropischer Xylariaceae/Taxonomía, Filogenia Y Biodiversidad Funcional De Las Xylariaceae Del Neotrópico) to Fundación Lillo, University of Buenos Aires and HZI.
Author information
Authors and Affiliations
Contributions
R.S.: methodology and analysis. C.L.: supervision, methodology, analysis, and writing—original draft preparation. J.A.J.: methodology. E.B.S.: methodology and writing—original draft preparation. M.S.: analysis, resources and writing—review and editing.
Corresponding author
Ethics declarations
All work on biological material presented in this paper, as well as its shipment and the long-term storage of cultures, was in accordance with a material transfer agreement between HZI and Fundacion Lillo, arising from the MINCyT/DAAD-PPP project “Taxonomía, Filogenia Y Biodiversidad Funcional De Las Xylariaceae Del Neotrópico.” Cultures were initially also stored at Fundación Lilo, where they did not survive. The copies that were maintained in Germany will be transferred to the culture collection of UBA (Buenos Aires) once ongoing work on the studied strains has been finished.
Ethics approval and consent to participate
N/A.
Consent for publication
All authors have agreed to the publication of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Section Editor: Marco Thines
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Characteristics of the nucleotide alignment and data matrix used for the molecular phylogenetic can be found in supplementary tables S1-S3. Single locus phylogenetic inferences can be found in Figure S1–S4. UV-Vis and mass spectrometric traces of the examined compounds can be found in Supplementary Figures S5–S15.
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Lambert, C., Schiefelbein, R., Jaimez, J.A. et al. Studies on Argentine Phylacia species (Hypoxylaceae) using a polythetic taxonomic approach. Mycol Progress 22, 27 (2023). https://doi.org/10.1007/s11557-023-01875-8
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11557-023-01875-8